Transcriptome analysis of female western flower thrips, Frankliniella occidentalis, exhibiting neo-panoistic ovarian development

The western flower thrips, Frankliniella occidentalis, is one of the most devastating insect pests with explosive reproductive potential. However, its reproductive physiological processes are not well understood. This study reports the ovarian development and associated transcriptomes of F. occidentalis. Each ovary consisted of four ovarioles, each of which contained a maximum of nine follicles in the vitellarium. The germarium consisted of several dividing cells forming a germ cell cluster, presumably consisting of oocytes and nurse cells. The nurse cells were restricted to the germarium while the subsequent follicles did not possess nurse cells or a nutritive cord, supporting the neo-panoistic ovariole usually found in thysanopteran insects. Oocyte development was completed 72 h after adult emergence (AAE). Transcriptome analysis was performed at mid (36 h AAE) and late (60 h AAE) ovarian developmental stages using RNA sequencing (RNASeq) technology. More than 120 million reads per replication were matched to ≈ 15,000 F. occidentalis genes. Almost 500 genes were differentially expressed at each of the mid and late ovarian developmental stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these differentially expressed genes (DEGs) were associated with metabolic pathways along with protein and nucleic acid biosynthesis. In both ovarian developmental stages, vitellogenin, mucin, and chorion genes were highly (> 8-fold) expressed. Endocrine signals associated with ovarian development were further investigated from the DEGs. Insulin and juvenile hormone signals were upregulated only at 36 h AAE, whereas the ecdysteroid signal was highly maintained at 60 h AAE. This study reports the transcriptome associated with the ovarian development of F. occidentalis, which possesses a neo-panoistic ovariole.

Introduction
The western flower thrip, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is one of the most devastating insect pests to many horticultural crops, especially those in greenhouses [1]. Both the larval and adult stages cause damage to plants by directly feeding on leaves or flowers. Especially, adults transmit plant viruses including tomato spotted wilt virus (TSWV) [2]. TSWV infection becomes serious and causes massive economic loss in hot pepper production in Korea [3]. This pest, originally native to North America, has spread to more than 60 countries since the late 1970s, including Canada, Australia, the United Kingdom, and far East Asian countries [4]. Various techniques such as chemical insecticides, entomopathogens, and pheromone traps have been applied to control F. occidentalis without satisfactory efficacy due to the insect's specific hiding behavior and insecticide resistance [5]. The thrips exhibits arrhenotokous parthenogenesis, with females developing from fertilized eggs and males from unfertilized eggs [6]. A brief immature period less than 10 days along with this various reproductive modes allow the thrips to rapidly build up the field populations during crop cultivating periods and so frequently leads to outbreaks beyond economic injury level [6]. Along with high reproductive potential, this type of mating behavior contributes to a rapid population increase and the development of insecticide resistance [7]. However, the molecular processes underlying reproduction and its regulation in this species remain unclear.
To investigate the physiological processes of the ovarian development of F. occidentalis, transcriptome analysis is useful for understanding the expressed genes associated with reproduction. A draft genome (415.8 Mb) of F. occidentalis was sequenced and its 16,859 genes were annotated into different functional categories including chemosensory receptors, detoxification, salivary gland, immunity, and development [8]. This suggests that RNA sequencing (RNASeq) analysis would be highly validated by this genomic information.
To identify the genes associated with ovarian development, this study investigated the ovarian development of F. occidentalis after adult emergence. After determining the mid and late ovarian developmental stages, transcriptomes were assessed using the NovaSeq 6000 platform. Subsequent differentially expressed gene (DEG) analysis in different developmental stages of female adults predicted the genes associated with ovarian development.

Thrip rearing
F. occidentalis adults were obtained from Bio Utility, Inc. (Andong, Korea) and reared in a laboratory under conditions of 27 ± 1˚C constant temperature, a 16:8 h (light:dark) photoperiod, and relative humidity of 60 ± 5%. The insects were reared on sprouted bean seed kernels.

Dissection of ovaries and microscopic observation
Different ages of female western flower thrips were dissected in 1 × phosphate-buffered saline (PBS) under a stereomicroscope at 30× magnification. PBS was prepared with 100 mM phosphate buffer containing 0.7% NaCl (pH 7.4). The ovaries were pulled from the abdominal tip and fixed with 3.7% paraformaldehyde in a wet chamber under darkness at room temperature (RT) for 60 min. After washing three times with 1 × PBS, the cells in the ovarioles were permeabilized with 0.2% Triton X-100 in 1 × PBS at RT for 20 min. The cells were then washed three times and blocked with 5% skim milk (MB cell, Seoul, Korea) in 1 × PBS at RT for 60 min. After washing three times, the cells were incubated with DAPI (4 0 ,6-diamidino-2-phenylindole, 1 mg/mL) diluted 1,000 times in PBS at RT for 2 min for nuclear staining. After washing three times, the ovarian cells were observed under a fluorescence microscope (DM2500, Leica, Wetzlar, Germany) at 200× magnification.

RNA extraction and RNASeq analysis
Total RNAs were extracted from the whole bodies of female F. occidentalis at different ages (0, 36, and 60 h after adult emergence). Three independent samples were used for three replications at each age. Each sample consisted of 50 females. RNA extraction was performed using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. An RNA library was generated using TruSeq Stranded Total RNA with Ribo-Zero H/M/ R_Gold (Illumina, San Diego, CA, USA). RNA sequencing was performed on the NovaSeq 6000 platform (Illumina) from Macrogen (Seoul, Korea). The RNA sequence was trimmed using CLC Workbench (QIAgen, Hilden, Germany). To calculate relative transcript accumulation, reads per kilobase per million (RPKM) mappable reads of the F. occidentalis genome (GenBank accession number: GCF_000697945.2) were estimated using CLC Workbench based on a template of Focc_2.1 version with a trimmed sequence of more than 50 bp.

Bioinformatics
DEGs were selected based on a fold change of � 2.0 and a P-value of < 0.05 with three biological replicates by comparing RPKM values at 36 h or 60 h after adult emergence (AAE) to those at 0 h AAE. KEGG pathway analysis was performed to test the statistical enrichment of DEGs using the KEGG mapper (https://www.kegg.jp/kegg/) by converting the National Center for Biotechnology Information (NCBI) Gene ID to a KEGG ID through the convert ID tool of KEGG mapper.

RT-qPCR
After RNA extraction, RNA was resuspended in nuclease-free water and quantified using a spectrophotometer (NanoDrop, Thermo Fisher Scientific, Wilmington, DE, USA). RNA (500 ng) was used for cDNA synthesis with RT PreMix (Intron Biotechnology, Seoul, Korea) containing oligo dT primers according to the manufacturer's instructions. All gene expression levels in this study were determined using a real-time polymerase chain reaction (PCR) machine (Step One Plus Real-Time PCR System, Applied Biosystem, Singapore) under the guidelines of [9]. Real-time PCR was conducted in a reaction volume of 20 μL containing 10 μL of Power SYBR Green PCR Master Mix (Thermo Scientific Korea), 3 μL of cDNA template (200 ng), and each 1 μL (10 pmol) of forward and reverse primers (S1 Table). After initial heat treatment at 95˚C for 2 min, qPCR was performed with 40 cycles of denaturation at 95˚C for 30 sec, annealing at 53~55˚C for 30 sec, and extension at 72˚C for 30 sec. The expression level of elongation factor-1 (EF-1, S1 Table) was used as a reference to normalize the target gene expression levels under different treatments. Quantitative analysis was performed using the comparative CT (2 -ΔΔCT ) method [10]. All experiments were independently replicated three times.

Statistical analysis
All the continuous variable data were subjected to a one-way analysis of variance using PROC GLM in the SAS program [11]. Means were compared with Duncan's multiple range test (DMRT) at type I error = 0.05.

Ovarian development in F. occidentalis
Dissection of 3-day-old female adults showed that a pair of ovaries contained eight ovarioles ( Fig 1A). The four ovarioles in each ovary were joined to a lateral oviduct and the two lateral oviducts were combined to the common oviduct. Each ovariole contained a string of follicles and was divided into a germarium and a vitellarium depending upon the presence of matured oocytes (Fig 1B). In the germarium, small cells were closely attached and formed a germ cell Ovariole structure divided into the germarium and the vitellarium. A total of 9 follicles are numbered from proximally to distally in the differential interference contrast ('DIC') picture. The white follicle represents chorionated oocytes. In the DAPI picture, each oocyte in the follicle is surrounded by follicular epithelium ('FE'). In the germarium, the germ cell cluster ('GCC') is located near to the oocyte ('OC'). (C) cluster. The vitellarium contained nine follicles, each of which was composed of oocytes and follicular epithelium without any nurse cells. The terminal follicles were usually chorionated.
Ovarian development occurred after adult emergence ( Fig 1C). The ovariole length began to extend just after adult emergence and reached a maximal size three days after emergence. Chorionated oocytes were visible after two days in some ovarioles. After three days, all the ovarioles had terminal chorionated oocytes, but some of the ovarioles lost them due to oviposition. This ovarian development pattern allowed us to determine three stages: early at 0 h, mid at 36 h, and late at 60 h after adult emergence.

Changes in the transcriptomes of female F. occidentalis adults during ovarian development
Total RNA was sequenced at three ovarian developmental stages of F. occidentalis. Each of the nine samples (= 3 stages × 3 replications) was sequenced from 10~13 Gb (Table 1). After trimming, 102~131 million reads in each sample were used to map to the F. occidentalis genome. With 71~82% mapping rates, the reads in each sample were matched to 14,042~14,521 genes among 16,859 predicted F. occidentalis genes. All the nine transcriptomes were deposited to GenBank with accession numbers of PRJNA833754.
When the transcriptomes of the three developmental stages were compared, they shared more than 95% (= 14,370/15,055) of the transcripts (Fig 2A). Forty-nine unique genes expressed at 36 h AAE were classified according to structure, gene regulation, and cell cycle along with several uncharacterized genes (S2 Table). However, their expression levels were extremely low at 0.0007~0.0468 RPKM. Forty-seven unique genes expressed at 60 h AAE were classified according to structure, protein-processing, and gene regulation along with several uncharacterized genes (S3 Table). Their expression levels were relatively high at 1.0016~1.6941 RPKM. However, this unique gene analysis did not identify genes apparently associated with oogenesis.
Ovarian development with female age. The ovariole length represents the germarium and vitellarium. Chorionated oocytes were counted per ovary. Each measurement used individual thrips and was replicated 5 times.
https://doi.org/10.1371/journal.pone.0272399.g001  4 Trimmed by CLC Workbench (QIAgen, Hilden, Germany). 5 Mapping to the F. occidentalis genome (GenBank accession number: GCF_000697945.2). 6 Total number of annotated genes was 16,859.  Table 1 To identify F. occidentalis genes associated with ovarian development, a differentially expressed gene (DEG) analysis was performed (Fig 2B). At 36 h AAE, 473 transcripts showed more than 2-fold increases in gene expression levels compared to those at 0 h AAE. At 60 h AAE, 443 transcripts showed more than 2-fold increases in gene expression levels compared to those at 0 h AAE. Only 19 transcripts showed changes between 36 h and 60 h AAE.
To characterize the DEGs at the mid (36 h AAE) and late (60 h AAE) ovarian developmental stages, their gene functions were predicted using KEGG analysis (Fig 3). Both DEGs were assigned to 52 KEGG functional categories but they did not exactly overlap. The DEGs of each developmental stage were assigned to 49 KEGG categories with three different missing categories. DEGs at 36 h AAE did not include the three categories of #21 (FoxO signaling pathway), #42 (protein export), and #50 (N-glycan biosynthesis), whereas the DEGs at 60 h AAE did not include the three categories of #17 (fatty acid biosynthesis), #30 (lysine degradation), and #39 (pentose and glucuronate interconversion). Among 46 common KEGG categories, most DEGs were classified into the metabolic pathway category (#33) in both developmental stages. The other major (> 10 DEGs) categories, biosynthesis of cofactors (#8) and lysosome (#31), were common in both developmental stages. However, the 36 h AAE samples had more DEGs in the inositol phosphate metabolism category (#28) than the 60 h AAE samples. In contrast, the glycan biosynthesis (#35) and purine metabolism (#44) categories had more DEGs at 60 h AAE than at 36 h AAE. The KEGG analysis suggested the upregulation of metabolic pathways associated with nucleic acid biosynthesis during ovarian development.

Expression profiles of egg proteins during ovarian development
To identify the specific genes associated with oogenesis in F. occidentalis, we selected genes that were highly expressed more than 8-fold at 36 h AAE or 60 h AAE compared to expression levels at 0 h AAE ( Table 2). The selected 99 genes were subdivided into structure, protein processing, lipid metabolism, gene regulation, and others. The structural protein category included typical egg proteins such as vitellogenin, chorion protein, mucin, and yellow melanization protein. In contrast, the highly suppressed genes at these stages included 37 genes (S4 Table). Especially, larval cuticular protein genes were included in the suppressed gene category.
The expression patterns of representative egg proteins during adult development were further analyzed (Fig 4). As expected, RNASeq analysis found that vitellogenin, chorion protein, mucin, and yellow genes were highly expressed in the mid and late ovarian developmental stages ( Fig 4A). However, there was little or no difference in the expression levels between the two developmental stages (36 h AAE and 60 h AAE). These transcript level profiles were further assessed by RT-qPCR with additional development stages (Fig 4B). The expression levels measured by RT-qPCR were mostly consistent with the expression profiles measured by RNA-Seq. However, RT-qPCR analysis indicated that the mucin and yellow genes were induced earlier than 36 h AAE. It also showed their expression patterns in late ovarian development after 60 h AAE, in which vitellogenin and mucin maintained the induced levels, whereas the expression levels of chorion protein and yellow significantly decreased.

Expression profiles of genes associated with endocrine signals during ovarian development
Juvenile hormone (JH), ecdysteroid, and insulin-like peptide (ILP) are well-known endocrine mediators during insect reproduction [12]. Genes associated with these endocrine signals were selected from the transcriptomes (Table 3). JH acid methyltransferase (JHAMT), JH esterase/ JH epoxide hydrolase, and Met involved in JH synthesis, JH degradation, and the JH receptor,   respectively, were identified. The RNASeq analysis showed that JHAMT was highly upregulated at 36 h AAE, whereas Met expression levels did not change during adult development ( Fig 5A). As ecdysteroid signaling genes, Shade and EcR were found in the adult transcriptomes. The expression levels of Shade were upregulated at mid and late ovarian development, during which EcR expression was slightly decreased. Insulin-like peptides and receptors were included in the adult transcriptomes. ILP was highly upregulated at 36 h AAE, whereas InR expression levels decreased during adult development. The transcriptome profiles related to endocrine signals were further supported by RT-qPCR analysis (Fig 5B). The RT-qPCR analysis assessed at several time points after adult emergence showed the upregulation of these endocrine signals during ovarian development. Especially, JH and insulin signals were more upregulated at 36 h AAE than at 60 h AAE, whereas the ecdysteroid signal was more upregulated at the late developmental stage.

Discussion
Various reproductive modes are observed in Thysanoptera. In F. occidentalis, a female produces progeny in bisexual or asexual arrhenotokous reproduction [13]. In arrhenotokous reproduction in the situation of few males (e.g., overwintering population), virgin females produce only male offspring [6]. When their sons are sexually mature, the females undergo bisexual reproduction with their sons and produce female-biased offspring. All thrips including F. occidentalis exhibit oviparous reproduction, in which females in either the asexual or bisexual mode undergo oogenesis in their ovaries. However, little is known about ovarian development in F. occidentalis. The current study analyzed the internal reproductive organ structure of F. occidentalis to assess the developing oocytes. Based on the temporal developmental pattern, RNASeq analysis was performed at early, mid, and late stages to determine the specific genes associated with ovarian development. The ovaries and associated internal reproductive organs of F. occidentalis were observed. Eight ovarioles from a pair of ovaries contained follicles, the end of which contained chorionated oocytes. Each follicle was composed of oocytes and follicular epithelium. The distal germarium contained a germ cell cluster, presumably consisting of interconnected nurse cells and oocytes. The ovarioles of insects are categorized into panoistic and meroistic types, in which the latter type is subdivided into polytrophic and telotrophic groups [14]. The panoistic type is considered to be the most ancestral because of its deficiency in transforming oogonia into nurse cells [15]. The polytrophic meroistic ovary has evolved from the panoistic ovary through the differentiation of nurse cells, and finally, the telotrophic meroistic type is derived from the polytrophic meroistic type by the restriction of nurse cells to the germarium. A deviation from the typical ovariole types is observed in Thysanoptera, in which the germ cell cluster is formed as seen in the ovary of the terebrantian thrip, Purthenothrips drucenae [16], suggesting that the panoistic follicles resulted from the secondary loss of nurse cells from the germ cell cluster. Stys et al. [14] called this type of ovary "neo-panoistic." Thus, the thysanopteran ovary provided new insight into the evolution of insect ovaries. Later, tubuliferan thrips also showed germ cell clusters, indicating that the neo-panoistic ovariole-type prevailed in Thysanoptera [17]. This was supported by our current study using F. occidentalis ovarioles, which did not have nurse cells in the follicles, while a germ cell cluster was found in the germarium. The ovaries of F. occidentalis grew just after adult emergence. During this period, the ovariole size increased along with oocyte development, and the final follicles in each ovariole had chorionated oocytes. Oogenesis is a sequential process consisting of previtellogenic development, vitellogenesis, and choriogenesis [18]. Previtellogenic development occurs in the germarium at the distal part of each ovariole and forms oocytes from the oogonial stem cells by mitosis and meiosis. Vitellogenesis is the process of accumulating vitellogenin (Vg) and other biomaterials into growing oocytes. After oocytes are fully grown, they are coated with chorion proteins secreted from the follicular epithelium to become eggs at the proximal part of the ovarioles. These eggs are then ovulated to the oviducts and fertilized just before oviposition. This ovarian developmental scenario suggests a sequence of oogenesis events in the neopanoistic ovariole of F. occidentalis. First, primary oocytes may be produced from germ cell clusters in the germarium. Second, the oocytes grow in size by accumulating Vg during vitellogenesis. Finally, the follicular epithelium forms the chorion of the fully grown oocytes during choriogenesis. Our transcriptome analysis supported the oogenesis processes by providing expression profiles of Vg, chorion proteins, mucin, and yellow genes during oogenesis.
RNASeq analysis used the NovaSeq platform, which sequenced more than 100 million reads per sample and resulted in more than 80% gene mapping rates. The first draft genome of F. occidentalis was reported and 16,859 genes were annotated [8]. Our RNASeq data from three ovarian developmental stages were mapped to 15,083 genes. Most of the mapped genes were shared among the three ovarian developmental stages. DEG analysis identified 473 and 443 DEGs in the mid and late stages, respectively. These DEGs were associated with metabolic pathways, especially related to nucleic acid biosynthesis and cofactor and amino acid biosynthetic pathways. The findings suggest that ovarian development in F. occidentalis requires a massive supply of raw materials such as nucleic acids and proteins.
Egg proteins were selected from the transcriptomes of the ovarian developmental stages. These genes represented highly expressed genes because they were increased more than 8-fold compared to the early ovarian developmental stage. The genes included mucin and yellow in addition to the well-known Vg and chorion protein genes. Mucins are high molecular and heavily glycosylated proteins with tandem repeats of identical or highly similar sequences rich in Ser, Thr, and Pro [19]. In insects, intestinal mucin is a major protein in the midgut peritrophic membrane, which facilitates the digestive process as well as protects the gut epithelium from microbial infections [20]. Salivary gland mucin might modulate the lubrication of insect mouthparts or defend plant attachment by inducing plant cell death through the formation of salivary sheaths [21,22]. A specific mucin protein is known to be associated with the formation of eggshells in Nilaparvapa lugens and Spodoptera exigua [23,24]. In our current study, the mucin gene highly expressed during F. occidentalis ovarian development suggests its function in chorion formation. Yellow and related major royal jelly protein (MRJP)-like proteins are widely found in insect genomes and these genes are classified into ten clades including Yellowb, -c, -d/e3, -e, -f, -g/g2, -h, -y, -x and MRJP-like protein [25]. In the Aedes albopictus mosquito, Yellow-g and Yellow-g2 are localized in the exochorion and outer endochorion, where they mediate darkening processes to physically strengthen the chorions [26]. Thus, the high expression of the yellow gene during F. occidentalis ovarian development suggests its function in chorion formation.
Transcriptome analysis also showed the expression profiles of genes associated with endocrine signals during F. occidentalis ovarian development. In insects, different endocrine signals are associated with ovarian development. JH is a sesquiterpenoid that mediates a status quo effect during the immature stage to prevent precocious metamorphosis. However, in adults, it stimulates ovarian development as a gonadotropin in various insects [27]. JH directly stimulates Vg biosynthesis in some insects and facilitates Vg uptake by growing oocytes by inducing follicular patency [28]. In mosquito females, 20-hydroxyecdysone acts as a gonadotropin [29]. ILPs are known to mediate ovarian development by stimulating oogonial proliferation to produce oocytes in the stem cell niche located in the germarium of the distal ovariole [30]. In F. occidentalis, JH and ecdysteroid play crucial roles in mediating metamorphosis. Krüppel homolog 1 (Kr-h1) and Broad (Br) are transcription factors leading to larval and pupal characteristics under JH and ecdysteroid hormones, respectively [31,32]. In F. occidentalis, Kr-h1 mRNA levels were high in the embryonic stage, remained at a moderate level in the larval and prepupal stages, and were low in the pupal stage. In contrast, Br mRNA levels were moderate in the embryonic stage and high at the larva-pupa transition stage. Except for Br expression in the embryonic stage, these two gene expression patterns followed the corresponding profiles of holometamorphic insects [33]. Furthermore, the adult specifier, E93, expression increased during immature development and its inhibition prevented adult metamorphosis [34]. However, little is known about JH and ecdysteroid mediation in oogenesis in F. occidentalis. Our current transcriptome analysis during ovarian development suggests that these endocrine signals play crucial roles in mediating F. occidentalis oogenesis based on their expression profiles. Increases in ILP and JHAMT expression at the mid ovarian developmental stage suggest their mediation of previtellogenesis by providing new oocytes from stem cells and vitellogenesis by stimulating Vg synthesis and uptake. Maintaining high levels of Shade expression suggest a high level of ecdysteroid during ovarian development, which may stimulate metabolic pathways, especially protein and nucleic acid biosynthesis, in addition to stimulating Vg synthesis with the cooperation of JH.
This study reports the comparative transcriptomes of F. occidentalis during different stages of ovarian development. Although the transcriptome analyses do not completely represent the protein expression profiles, they gave us valuable insights on the thrips reproduction. The results suggest an increase in metabolic pathways along with protein and nucleic acid biosynthesis. The high upregulation of egg proteins such as Vg, chorion protein, and sclerotizing agents during choriogenesis was also found. Finally, JH, ecdysteroid, and insulin signals may play crucial roles in mediating F. occidentalis oogenesis. A recent study showed that prostaglandin mediates oocyte devilment in early and late stages in addition to the endocrine signals [35]. This suggests the oogenesis of F. occidentalis would be a model system for an integrative analysis of endocrine signals mediating different reproductive processes of previtellogenesis, vitellogenesis, and choriogenesis.


Higher-order structures of the foot-and-mouth disease virus RNA-dependent RNA polymerase required for genome replication

Replication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are thought to provide a favourable environment for replication to occur, concentrating essential viral structural and nonstructural components, as well as protecting these components from host-cell pathogen recognition and innate immune responses. However, the details of the molecular interactions and dynamics within these structures is very limited. One of the key components of the replication machinery is the RNA-dependent RNA polymerase, RdRp. This enzyme has been shown to form higher-order fibrils in vitro. Here, using the RdRp from foot-and-mouth disease virus (termed 3Dpol), we report fibril structures, solved at ~7-9 Å resolution by cryo-EM, revealing multiple conformations of a flexible assembly. Fitting high-resolution coordinates led to the definition of potential intermolecular interactions. We employed mutagenesis using a sub-genomic replicon system to probe the importance of these interactions for replication. We use these data to propose models for the role of higher-order 3Dpol complexes as a dynamic scaffold within which RNA replication can occur.

T he replication of genomic material is a key stage of the lifecycle of all viruses. Some of the molecular details of replication are well defined as a result of elegant in vitro analyses. It has also been demonstrated that for many viruses, replication occurs in macromolecular complexes involving both viral and cellular components [1][2][3][4][5][6] . However, the molecular details of the architecture of replication complexes remains unknown and thus we do not fully understand how proteins and viral genetic material are associated during the replication process or how dynamic rearrangements of higher-order assemblies may regulate RNA synthesis during viral replication.
The genomes of positive-sense RNA viruses act directly as a messenger RNA and hence are translated immediately after infection. For picornaviruses such as poliovirus (PV), rhinovirus and foot-and-mouth disease virus (FMDV), proteolytic processing of the viral polyprotein ultimately releases the nonstructural proteins involved in genome replication. A key protein is the RNA-dependent RNA polymerase, RdRp, termed 3D pol . This enzyme (in complex with other viral proteins e.g., 2C, 3A, and 3CD and a replication primer protein termed 3B) produces negative-strand genomic intermediates, from which new positive strands are synthesised [7][8][9][10] .
Biochemical and structural studies have yielded a very good understanding of the stages involved in each step of nucleotide incorporation 11 . In common with other DNA and RNA polymerases, the structure of the picornavirus 3D pol resembles a cupped right hand, with three defined subdomains, termed thumb, fingers and palm 12,13 . The finger subdomains are extended in picornaviral 3D pol structures, when compared to many other polymerases, and contact the tip of the thumb subdomain. This contact is important for maintaining polymerase structure whilst the geometry of the fingers against the thumb form part of the NTP entry channel. The single-stranded template RNA enters 3D pol through a channel at the face distal to the active site and takes a right-angle turn at the palm active site when the producttemplate duplex is formed. This emerges from the large front opening [14][15][16][17][18][19] . There is, however, no strand separation system identified from polymerase biochemical-structural data alone, but such a mechanism must presumably exist to prevent the formation of a stable RNA duplex.
In contrast to its catalytic role in replication, the non-catalytic roles of 3D pol are less defined. It forms part of a larger precursor 3CD, and is essential for RNA replication via genomic cyclisation 9,20,21 . Previous studies have also suggested that 3D pol (or larger precursors thereof) has an essential cis-acting role in replication (i.e., a structural function which needs to be provided from the replicating genome), which is distinct from a separate trans-acting role (i.e., the enzymatic function of the polymerase that can be supplied by a separate genome) 19,22 . Dimers and higher-order oligomers of RdRp molecules have been reported in a number of positive-sense RNA viruses [23][24][25][26][27] . Furthermore, the 3D pol enzyme of both PV and FMDV has been previously shown to form higher-order fibrillar structures 25,[28][29][30][31] . PV 3D pol has also been demonstrated to form planar lattices and higher-order structures have been visualised within infected cells 25 . It has therefore been postulated that the higher-order structures act as a replication scaffold; however, no RNA has been shown within the PV 3D pol structures and furthermore, these were demonstrated to form in vitro in the absence of RNA 28,30,31 . In contrast, the FMDV fibrillar structures were only detected in vitro in the presence of RNA. Indeed, our previous studies showed that primer, RNA template and nucleotides were required for fibril formation, thus suggesting that they could be part of an active replication complex 29 .
Using immunofluorescent confocal and electron microscopy, FMDV has been shown to dysregulate Golgi and ER-derived membranes in a similar way to PV 1,5,6,32,33 , but distinct membrane-bound replication complexes comprised of viral RNA, structural and nonstructural proteins, and host-cell proteins have yet to be identified for FMDV. Although both viruses are members of the Picornaviridae, the Aphthovirus FMDV has several unique features that distinguish it from other members of the family including an extended 5′ untranslated region and triplication of the primer encoding region.
Picornaviruses are responsible for several important human and animal diseases. For example, FMDV, the causative agent of foot-and-mouth disease, is a significant economic problem, such as occurred in the UK in 2001 (where economic losses to farming and tourism were estimated at~£15 billion at current value) 34,35 . Therefore, a more complete understanding of the composition of the replication machinery is important for therapeutic development. Here, we have trapped the products of a replication assay by crosslinking and thus captured higher-order structures in a range of conformations. Using cryo-electron microscopy, we have calculated the structures of 3D pol fibrils in 9 conformations at 7-9 Å resolution, with some of these occurring within the same fibril. We also probed the importance of 3D pol -3D pol interfaces within the fibril by mutagenesis and we use these data to build a model of conformational changes in higher-order assemblies of 3D pol that may be important in replication.

Results
Wild-type 3D pol can catalyse incorporation of UTP into nascent RNA. We first sought to demonstrate that the recombinant 3D pol protein was able to bind a synthetic template RNA and was catalytically active. We produced wild-type (WT) 3D pol in E. coli as previously described 15,29 , alongside 3D pol with mutations in the active site (3D pol -GNN) as a control. To assess the affinity of RNA binding by 3D pol , a fluorescence polarisation anisotropy (FPA) assay was performed. Samples of 3D pol -WT and 3D pol -GNN were titrated with FITC-labelled 13mer poly-A RNA (Fig. 1a), resulting in Kd values of WT and GNN of 5.83 ± 1.3 and 1.92 ± 0.67 μM, respectively (Fig. 1b). These data showed that both active and inactive enzymes were able to bind RNA in vitro, with the mutant demonstrating significantly higher affinity (p < 0.01).
The ability of the protein to incorporate [α-32 P] UTP into RNA, was assessed as described in 29 utilising a poly-A template and an oligo-d(T) primer, supplemented with a 5:1 ratio of UTP to [α-32 P] UTP. After incubation at 30°C, polymerase activity was assessed by dot-blot and quantified by scintillation counting (Fig. 2a, b). Normalised scintillation counts quantifying the dotblot samples incubated for 30 min (Fig. 2b) show a 25-fold greater incorporation of [α-32 P] UTP for WT compared to the enzymatically inactive 3D pol -GNN.
Formation of higher-order complexes. We have previously demonstrated the formation of higher-order fibrillar complexes of 3D pol by negative stain transmission electron microscopy (TEM) 29 . To investigate the assembly of the fibrils, we employed glutaraldehyde crosslinking both to trap higher-order oligomers and stabilise the fibrils. Samples of recombinant 3D pol were incubated at 30°C in the presence of glutaraldehyde and the RNA template-primer used for the activity assay described above. At time intervals, the reaction was stopped by the addition of Laemmli buffer. The samples were resolved by gradient SDS-PAGE and 3D pol -WT or 3D pol -GNN multimers detected by anti-3D pol immunoblot (Fig. 3a, c, respectively) and quantified by densitometry (Fig. 3b, d, respectively). An increase in oligomer formation over time was evident, which seemed to correlate predominantly to the addition of dimers. Higher-order oligomer formation appeared to be more rapid for 3D pol -GNN than WT, however, the differences were not statistically significant. Although higher-order oligomers were present in the absence of RNA, this did not correlate with the formation of fibrils. At later timepoints, complexes were too large to migrate into the gel (Fig. 3a, c).
Structural analysis of FMDV 3D pol fibrils. To investigate higher-order fibril structures in the presence of glutaraldehyde, samples of 3D pol -WT and 3D pol -GNN were taken at 30 min. Samples were prepared for TEM imaging using negative stain (Fig. 4a, b). This analysis showed that 3D pol -WT was able to form higher-order fibrillar structures, similar to those we reported previously 29 (Fig. 4a). Larger structures produced by 3D pol -GNN appeared as protein aggregates (Fig. 4b), similar to those occasionally found in samples of 3D pol -WT incubated in the absence of RNA, suggesting that the faster oligomerisation of 3D pol -GNN (shown in Fig. 3) may result in less ordered assembly. The fibrils were susceptible to RNase treatment (Fig. S1), in agreement with previous data 29 . We have previously reported the X-ray structure of a 3D pol bearing a mutation in the RNA exit channel 19 . We found this mutant to also be deficient in fibril formation, consistent with a deficiency in RNA binding (Fig. S1). The availability of fixed, stable 3D pol -WT fibrils led us to investigate their structure by cryo-EM.
To this end, 3D pol -WT fibrils were produced as above, prepared for cryo-EM and imaged on a Thermo-Fisher Titan Krios cryo-electron microscope. Fibril sections were picked and subjected to 2D class-averaging, which appeared to show variation in fibril diameters (Fig. 4c). Measurements from 1D projections of these 2D classes along the fibril axis confirmed this, showing a bimodal distribution of diameters that ranged betweeñ 20 nm and~23 nm. The modal diameters were 21.3 nm and 22.6 nm, indicating the presence of at least two forms of fibril (Fig. S2). These values correlate well with the size of fibrils previously measured from negative stain TEM images 29 .
Helical reconstruction with 3D classification was performed, separating the dataset into fifteen self-similar classes. In this analysis, nine classes gave reconstructions that exhibited sharp density. These were individually refined leading to the definition of fibril reconstructions with diameters that reflected the bimodal distribution seen in the 2D class averages. Of these nine conformations, two were defined as 'broad', B1 and B2corresponding to the 2D classes having a modal diameter of 22.6 nm. Seven reconstructions were classed as 'narrow'; N3-N9, corresponding to the larger group of fibrils having a modal diameter of 21.3 nm. The distinction between broad and narrow fibrils was made based on the helical symmetry of each group (see below). Reconstructions ranged in resolution between 7.3 Å and 9.5 Å ( Fig. 4d-g, Fig. S3, Table 1, Movie S1).
Each reconstruction was readily interpreted by rigid-body fitting of the crystal structure for FMDV 3D pol (PDB ID 2EC0) revealing that all the solved fibril conformations are right-handed 2-start helical assemblies, each comprising a lattice of repeating 3D pol dimers (Fig. 4h, i). Dimers are rotationally symmetric about their interface and each of the two protofilaments is a ribbon of 3D pol dimers that is therefore antiparallel. The nine conformations that were resolved through our analysis show that variation in fibril diameter is a consequence of the two protofilaments sliding and flexing relative to each other (Movie S1). Among these reconstructions, four distinct forms of fibril were identified. The presence of a bimodal distribution of fibril diameters reflects the presence of two helical symmetries. Broad fibrils were found to be 2-start helices having C2 symmetry about the helix axis. Protofilaments had between 9 and 9.2 dimeric subunits per helix turn. Narrow fibrils were not C2 symmetric and exhibited 2-start helical symmetry with between 8.4 and 8.6 3D pol dimers per helix turn in each protofilament ribbon. A notable difference between  the broad and narrow classes of fibrils was that narrow fibrils could also be described by a left-handed 1-start helix of 3D pol dimers having a twist of~-159°and an axial rise of 15-17 Å while the twofold symmetric, broad-fibrils could not (Table 1).
Within the narrow and broad classes, fibrils were found to form either tightly packed or open conformations. N3-N8 were tightly packed fibrils in which flexing of protofilaments resulted in a progressive tightening of packing from N3 to N8, with N8 being the most densely packed of the narrow fibril forms. N9 shows a rather different assembly with a longer axial rise and, although the fibril is narrower, a more open structure with large gaps between protofilaments. Similarly, B1 and B2 showed tight and open forms respectively, with B2 being narrower than B1 and having a longer axial rise per subunit. Three-dimensional classification did not distinguish more subtle variations for broad fibrils (similar to those seen in the tightly packed narrow fibril classes (N3-N8)), possibly a consequence of fewer broad fibrils being present in the dataset. In both N9 and B2 (the open fibril conformations) the spaces between protofilaments contained noisy density that was not accounted for by fitting the atomic model for 3D pol , we postulate that this may be RNA (see below). It is also important to note that the RNA entry and exit sites are not occluded (except by this putative RNA density).
Movie S1 shows morphing between fibril conformations, this is intended to illustrate the differences in these structures rather than model any conformational changes that may occur. To determine whether the fibril classes solved in our analysis represented discrete structures or continuously varying helices, we traced fibril image sections from each of the reconstructions back to their original micrograph (Fig. 5). We found that the narrow fibrils that contributed images to reconstructions N3-N8 varied between these forms along their length, suggesting that these reconstructions represent snapshots of a continuously flexing assembly. Fibrils were seen to switch conformation between tight and open forms. In most cases, changes in fibril conformation were marked by visible constrictions and the different forms presented as discrete regions of longer fibrils, rather than the continuous variation seen for the narrow/tight classes. Overall, these data suggest that narrow and broad forms were distinct from one another. Fibrils that appear to show both broad and narrow forms along their length were, however, noted and in these images pronounced discontinuities/constrictions were observed.
Inter-and intradimer interactions. As noted above, an inspection of our reconstructed density maps along with rigid-body fitting of high-resolution coordinates for 3D pol indicated that fibrils each comprise two protofilaments that are ribbons of dimers. Analysis of contacts and clashes between 3D pol monomers in our models confirms this. These data are also consistent with fibril formation by dimer addition as shown in Fig. 3. In all conformations, the dimer interfaces and dimer-dimer interfaces are similar, while different interfaces between protofilaments were seen in tight and open forms (Figs. 5a and 6a-e; Movie S2; Table S1). To describe the interactions between monomers within Fig. 3 Elongation of protein monomers into higher-order oligomers. Polymerase activity assay in the presence of glutaraldehyde, showing the formation of higher-order oligomers by representative Western blot of a polymerase activity assay of a 3D pol -WT and c 3D pol -GNN, b and d. Quantification of 3D pol higher-order oligomer formation of 3D pol -WT and 3D pol -GNN, respectively, over time by densitometry. Control reactions were included without primer (-P), without RNA template (-R), or without glutaraldehyde (-G) and analysed after 120 min. Values normalised to the average densitometry values (a.u) at each timepoint across three biological repeats using ImageJ. Approximate molecular weights are indicated, by comparison to Coomassie-stained gels. Comparisons between timepoints are within 3D pol -WT and 3D pol -GNN respectively (n = 3, ± SEM, analysed by two-way ANOVA, *p < 0.05, **p < 0.001). fibrils we use a notation incorporating the 'palm', 'fingers', and thumb' subdomain nomenclature, while the relative spatial organisation of monomers within the helix are denoted as follows. Where n represents a monomer and then n' is the twofold symmetry-related monomer within a single dimeric subunit (Fig. S4). Here, n + 1 represents the next monomer, on the opposite protofilament (and n' + 1 its partner within a dimer). For conformations N3-N9 the transformation from n to n + 1 and all subsequent increments is defined by the left-handed 1start helical parameters: a rotation of ∼-158°and an axial rise of ∼15-17 Å. For the C2 symmetric broad fibrils, which do not show 1-start helical symmetry, the transformation from n to n + 1 (and Fig. 4 Structure of 3D pol fibrils. Negative stain TEM of a sample from a polymerase activity assay in the presence of glutaraldehyde after 30-minute incubation. a 3D pol -WT forms visible fibrils, b 3D pol -GNN does not show any distinct fibril structure formation. Scale bar 100 nm. c 2D classification and averaging of fibril section images from cryo-EM data shows variation in fibril diameter. d-g Helical reconstruction led to the calculation of density maps for nine classes of fibril at 7.3-9.5 Å resolution, four representative maps are illustrated here coloured according to radius (see key below panel g). d The broadest fibril reconstruction; B1 had a diameter of 22.3 nm, C2 symmetry, a helical twist of 39 o and an axial rise per subunit of 27 Å. e Conformation B2 has a more open structure, is also C2 symmetric and has a diameter of 21.7 nm, twist of 40 o and rise of 31 Å. f N3 is a narrow (Ø = 21.5 nm) and tightly packed fibril which may also be described by a 1-start helical symmetry with a twist of -158.8 o and axial rise of 15 Å. g N9 is a narrow, open fibril structure having a diameter of 20 nm, twist of -158.6°and axial rise of 17.4 Å. h Fitting of the X-ray structure for 3D pol -WT in complex with template primer RNA and ATP (PDB: 2EC0) into each density map shows that these fibrils are two-start helical lattices comprising protofilaments of 3D pol dimers. i Protofilament one is coloured violet/thistle, while protofilament two is coloured teal/aquamarine, two shades are used in each protofilament to highlight how 3D pol monomers form ribbons of dimers. subsequent odd-numbered monomers) is simply a rotation of −180°. The transformation from n + 1 to n + 2 (and all subsequent even-numbered monomers) is a rotation of ∼-140°and an axial rise of~27-31 Å. Under this notation, for all fibril conformations, n + 2, 4, 6 etc. are successive monomers on the same protofilament as n, while n + 1, 3, 5 etc. are on the other. The dimer interface is between thumb subdomains, predominantly at the C-terminal helix α16 (residues 453-467) of n and n' (Fig. 6b). Dimers repeat along each protofilament ribbon with the primary interaction being between the thumb/fingertips of n and the palm sub-subdomain of n + 2 (and between the palm subdomain of n' and thumb/fingertips of n'+2; Fig. 6c). Interactions between protofilaments in each conformation are also twofold rotationally symmetric owing to the antiparallel nature of each protofilament. The interactions are not, however, conserved between fibril forms. In tight conformations, the contact interface is mainly within a loop at amino acid residues 9-17, between n and n'+9 or n'+7 for B1 and N3-N8, respectively (Fig. 6d). In conformation N8, there is an additional point of contact between n and n'+5 between V4 in strand β1 and K65 in the N-terminal loop of the palm subdomain. In open fibrils, the contacts/clashes between protofilaments are between n and n'+7 for B2, and n and n'+5 for N9. In both cases β1 strands in each monomer come together, possibly forming an extended antiparallel β-sheet (Fig. 6f).
Comparison of our structures for fibrils formed of FMDV 3D pol with previously published oligomeric forms of PV 3D pol shows that these molecules pack differently. X-ray crystallography studies have shown that PV 3D pol molecules form functional dimers via a strong interaction termed 'interface 1' between the C-terminal helix in the thumb subdomain, and the back face of the palm subdomain 23 . Furthermore, PV helical polymerase lattices form much larger (50 nm) fibrils, helical reconstructions of which have shown a 6-start helix of protofilaments formed of interface 1 dimers 31 . However, it should be noted that the PV 3D pol was untagged, whereas the FMDV 3D pol used here was C-terminally his tagged. 470 residues (of 476 excluding the tag) were modelled. Although density extends beyond this, it is not possible to determine the position of the tag.
The third interaction site (between protofilaments) is highly variable. Our fitting experiments led to the definition of contact interfaces for tight forms comprising four residues. The considerable flexing of protofilaments argues for a weak interaction at this site. The open forms show a more intriguing potential interaction -formation of an extended antiparallel β-sheet via hydrogen bonding between residues 4-8. Mutagenesis to target this interface would require disruption of secondary structure. For these reasons, we chose to focus our mutagenesis studies on those interactions that support protofilament formation.
To understand whether the intermolecular interactions above are important for viral replication we employed a replicon system 36,37 . We hypothesised that if the polymerase intermolecular interactions described here are biologically important, disruption of these interactions would impair function, resulting in reduced replicon replication. We therefore selected several residues from both the thumb-to-thumb interface (Y394, F398, R456, Y459, L460, V463, and C467) and the fingers/thumb-topalm interface (P23, V25, E324, Y346, D347, I420, P445, L449) for mutation. Selecting residues at random from both interfaces prevented biasing the outcome by focusing the mutagenesis on specific secondary structures or amino acid physical properties; however, it should be noted that all of the residues are conserved between FMDV isolates. Each of the selected residues was individually mutated to alanine in the context of a FMDV replicon in which the structural proteins were replaced by GFP. Measuring GFP expression therefore allows the replication competency of each mutation to be assessed. From each mutant replicon, RNA was generated by T7 transcription and transfected into BHK-21 cells. Replicons with 3D pol -WT and 3D pol -GNN were included as controls, the latter for the level of translation from the input RNA. RNA replication was monitored by GFP fluorescence, with replication shown as the number of GFP positive cells at 8 h post-transfection, analysed according to our established protocols 37 (Fig. 7a).
Of the fifteen amino acids mutated only five: F398A, R456A, Y459A, L460A, and C467A, significantly reduced FMDV replicon efficiency compared to the WT replicon. All of these residues were located within the thumb-to-thumb interaction (coloured dark magenta, Fig. 7b). All the other mutations tested showed levels of replication equivalent to a WT replicon (coloured orange Fig. 7b, c). This included two mutations (V463 A and V466A) and all of the mutations made at the fingers/thumb-to-palm interface. Overall, these data suggest that residues involved in dimer formation are important for FMDV replication, potentially through allowing the formation of intermolecular 3D pol -3D pol interactions within the FMDV replication complex.
Unassigned density within 3D reconstructions may represent RNA. Within each of our fibril reconstructions, density within the active site of individual 3D pol molecules was visible, however, this was very weak in comparison to the protein density. Nonetheless, the data suggest the presence of retained RNA at low occupancy within fibrils (Fig. 8, Movie S3). In the open conformations (reconstructions B2 and N9) there is an additional unassigned density that extends between two 3D pol molecules, bridging an opening in the fibril between protofilaments. The density is poorly resolved but unambiguously present, we suggest that this may represent RNA entering or extruded from the active sites.

Discussion
The RNA-dependent RNA polymerase, 3D pol , was one of the first proteins to be characterised both in PV 38 and in FMDV 39 and is the key enzyme in viral replication. Both PV and FMDV 3D pol have been shown to form higher-order structures in vitro in the form of fibrils 25,[28][29][30][31]40 that have been postulated to be part of viral replication complexes. It would be expected that replication complexes would be dynamic structures, and although there are structural data available for the PV fibrils these are at relatively Fig. 7 Replication of 3D pol mutants. Intramolecular contact residues were identified and were mutated to alanine within the 3D-encoding region of ptGFPexpressing subgenomic replicon. a Replicon RNA was transfected into BHK-21 cells and replication determined by measuring the levels of GFP expression in comparison to the level of translation (GNN). Residues that significantly reduced the level of GFP expression are highlighted in pink. b and c Location of the amino acid residues that were mutated in this analysis, those that are coloured orange did not significantly affect replicon efficiency, while those that are coloured dark magenta significantly reduced the levels of GFP expression and were located at the modelled 3D pol dimerisation interface only (PDB: 2EC0). Statistical analysis by unpaired two-tailed t test, ± SEM, **p < 0.001, ***p < 0.0001.  Fig. 4f), shows the presence of weak unassigned density in the active site of 3D pol that we attribute to RNA (white arrows). d, e the same view is presented as a transparent isosurface with PDB 2EC0 shown. The atomic model is shown with protein represented as a rainbow coloured ribbon diagram and RNA as sticks coloured mauve, to highlight the assignment of protein and RNA to cryo-EM density. c, f Conformation N9 also shows unassigned density in the active site as well as a finger of density linking two protofilaments (red rectangle) that may be RNA template or product extruded from the active site. low resolution, did not include any RNA and were thus unable to give insight into any dynamics 30,31 . Here, we report multiple conformations of the fibrils formed by FMDV 3D pol . Some assemblies exhibited multiple conformations along their length. Furthermore, we report the presence of additional density in fibril reconstructions that we attribute to RNA. We probed the interfaces by mutagenesis and are using these data to propose a model for the potential role of conformational changes in the fibril during genome replication.
We have here demonstrated that the recombinant 3D pol protein was able to bind RNA and was catalytically active, this led to the formation of fibrils. In contrast, two mutants that were deficient in replication did not form ordered products. We analysed the structures of the assembled fibril products of a polymerase activity assay after crosslinking. This provided a total of 9 different 3D pol reconstructions, all of which are at higher resolution and distinct from the previously reported structures of PV fibrils, in terms of both dimensions and the orientation of molecules within the helix. The dimerisation interface of the FMDV 3D pol molecules is novel and distinct from the dimerisation of PV 3D pol molecules. The formation of fibrils by dimer addition is also supported by the cross-linking data presented here. Mutations that affected the ability of the polymerase to transcribe replicon RNA were introduced within the dimerisation thumb-thumb interface. This contact region has been implicated in the formation of the PV fibrils and shown to be necessary in the production of virus, despite being distant from the active site 41 . In PV, a model has been described whereby RNA would travel through the active site between polymerase molecules; however, RNA was not detected 25,30 . In support of this model, density corresponding to RNA was detected in the FMDV fibril structures presented here. This density appears in the active site of all our reconstructions and is also seen linking individual molecules in the open fibril conformations.
The formation of a classical intracellular, membrane-associated replication complex for FMDV has yet to be demonstrated within infected cells. One could speculate, in the context of a cellular replication complex, that the polymerase molecules could coat newly synthesised negative-sense intermediates and prevent the formation of double-stranded RNA, a potent innate immune response trigger. Avoiding double-stranded RNA formation would also allow for a more energetically-favourable replication process. By coating RNA by the addition of dimers, helical fibrils similar to those described here could be generated. The concept of 3D pol acting in a similar way to the N protein of negative-sense viruses or the coat protein of helical viruses such as tobacco mosaic virus is intriguing.
The structures described here could alternatively represent trapped conformers of the active replication machinery within the functional complex. The different fibril conformations observed could represent different stages of replication. Although there is some density for RNA in the active site of 3D pol molecules within the fibrils, a model where RNA is looping out from the body of a dynamic fibril would be consistent with the relatively weak RNA density seen overall and with the observation that the fibrils are sensitive to RNaseA. However, it is also tempting to speculate that the density seen between 3D pol molecules in the open N9 and B2 conformations correlates with newly synthesised RNA being extruded from the active site. The 9 fibril structures described here are broadly representative of 4 types: narrow, broad, tight and open. Data for the narrow fibrils were consistent with these being a continuous, flexing assembly, with the broad fibrils a separate, distinct class. Given the breadth of fibril morphology shown here, it is therefore possible that both models described above are correct. Some of the fibrils could form by the simple addition and dissociation of 3D pol subunits and act to coat newly formed RNA transcripts. Others could form part of a dynamic replication complex. Different pools of 3D pol molecules involved in replication would be consistent with the models we proposed as a result of complementation experiments 19 . Correlative light-electron microscopy studies in replicon-containing cells are underway in order to further elucidate the formation and dynamics of complexes within cells and to establish if this and other nonstructural proteins, such as 3CD, 3A and 3B, are able to interact with the FMDV 3D pol fibrils.

Methods
Expression and purification of his-tagged 3D pol . WT C-terminal His-tagged 3D pol was expressed from vector pET-28a (a kind gift from Esteban Domingo, Centro De Biologia Molecular Severo Ochoa, Madrid, Spain) 42 in Codon plus (+) BL21 using methods described previously 15 . For lysis, the cells were suspended in 20 mM Tris-HCl [pH 8] lysis buffer with 20 mg/ml lysozyme before sonication as described previously 15 . Lysates were clarified and the supernatant filtered through 0.22 µm filters and subjected to nickel affinity chromatography using 1 ml HisTrap HP columns (GE Healthcare). Elution was carried out via a 25-500 mM imidazole gradient in 50 mM Tris [pH 8], 500 mM NaCl buffer. Fractions containing the protein were pooled, dialyzed and stored in aliquots at -20°C as described previously 19 . A replication-deficient enzyme (termed GNN which includes the active site mutations D338N and D339N 19 were expressed and purified in the same way and the mutations confirmed by mass spectrometry. 3D pol RNA-binding by fluorescence polarisation anisotropy. The ability of FMDV 3D pol to bind to RNA was examined using fluorescence polarisation anisotropy (FPA) adapted from published protocols 43 . FPA measures the tumbling rate of a fluorescently labelled molecule. After excitation by polarised light, the excited fluorophore emits light with the degree of polarisation inversely proportional to the tumbling rate of the fluorescent molecule in the solution. Therefore, the smaller the molecule, the higher the tumbling rate resulting in the depolarisation of the emitted light as the fluorophore reorients in solution during the lifetime of the excitation. FPA was undertaken in a black opaque 384-well format (Perkin Elmer). 20 µl of RNA binding buffer (20 mM Tris-HCl (pH 8), 100 mM NaCl, 0.01 % Triton X-100) was added to each well followed by a serial dilution of purified 3D pol or mutant 3D pol -GNN. 20 µl of 20 nM 3′FITC-labelled 13mer poly-A (Dharmacon) (kind gift from Thomas Edwards, University of Leeds) was overlaid into each well and samples incubated at room temperature for 1 h. Control samples that contained no fluorescent RNA to establish background polarisation were included. Polarisation was measured using a Tecan plate reader with a 480nm excitation filter and 530 nm S and P channel emission filters.
3D pol Activity Assay. Activity of purified FMDV 3D pol was assessed by the ability of the protein to incorporate [α-32 P] UTP into nascent RNA transcripts, as previously described 29,44 . The final concentration of 3D pol that was included in the assays was 18.52 µM. Reactions were stopped by the addition of 1.5 µl 500 mM EDTA at the indicated times. For scintillation, 2 µl of each reaction was blotted onto filter paper (Whatmann) and allowed to dry prior to being washed as described previously 44 . The dry filter paper was exposed to film for imaging before the individual dots on the filter paper were excised and scintillated with the addition of 3 ml scintillation fluid (Perkin Elmer). Radioactive counts were measured as scintillation counts per minute.
For glutaraldehyde crosslinking, 3D pol activity assays, substituting [α-32 P] UTP with 4 mM UTP were employed. Samples were incubated with 5 mM glutaraldehyde at 30°C for increasing time periods and reactions stopped by the addition of 30 µL 2x Laemmli buffer. The samples were denatured and analysed by gradient SDS-PAGE (12-4% resolving gels, and 4% stacking gel). Immunoblotting was performed as described previously 45 with primary rabbit anti-3D 397 polyclonal antibody (kind gift from Francisco Sobrino, Centro De Biologia Molecular Severo Ochoa, Madrid, Spain) and detected with anti-rabbit HRP (Sigma-Aldrich).
Transmission electron microscopy (TEM). Carbon-coated copper grids were prepared by glow discharge at 25 mA for 30 seconds. The activity assay master mix was prepared as described for the crosslinking assay. Samples were incubated for 30 min at 30°C and then immediately 10 µl was adsorbed onto carbon-coated copper EM grids for 1 min. The excess sample was removed and the grids washed twice with 10 µl dH 2 O for 5 seconds prior to staining with 10 µl 2% uranyl acetate for 1 min, the excess was then removed with filter paper. TEM was analysed on the Jeol 1400 (Jeol USA Inc.) at 30,000 x magnification.
Cryo-electron microscopy (Cryo-EM). Activity assay master mix was prepared as described above and incubated at 30°C for 30 min. 10 µl of the master mix was loaded on a C-flat (protochips) CF1.2/1.3-4 C grid (1.2 µm holes) that had been prepared by addition of a continuous carbon support film and glow discharged at 25 mA for 1 min. The grids were blotted for 6 seconds at blot force 10, using an FEI Vitrobot Mark IV in chamber conditions of 4°C and 95% relative humidity. These were screened and initial analysis performed using a JEOL 2200 TEM equipped with a Direct Electron DE20 camera, optimised grids were then imaged on a Thermo-Fisher Titan Krios in the Astbury Biostructure Laboratory using a Falcon III detector in integrating mode operated at 40 frames per second. Data were collected at nominal 75,000 x magnification, giving a sampling frequency of 1.065 Å/pixel. Two-second exposures were collected at a dose rate of 54 e − /Å 2 /second and a defocus range of −1.5 to 2 microns.
Image processing. Preliminary analysis of a small dataset collected on a JEOL 2200 was performed using Relion 2.0.3. A single 2D class average was used to generate a starting model using SPIDER. Initial estimates of approximate helical parameters were derived by taking measurements from 2D class averages (twist = 40°and rise per subunit of 29 Å). Calculation of an initial helical reconstruction was performed giving rise to an interpretable density map with twist = 42.4°and rise of 30 Å. Inspection of this preliminary map revealed a two-start helical assembly (Fig. S5).
Collection of the substantially larger dataset was undertaken at the Astbury BioStructure Laboratory. 5417 micrographs were recorded from which 266,263 images of fibril sections were selected. Motion correction was performed using Motioncor2 with dose-weighting 46 . CTF estimation was performed using GCTF 47 . Helical reconstruction was performed using Relion 3.0 followed by 3.1 48,49 . Following 2D classification the dataset was reduced to 247,623 fibril section images that had contributed to sharp class averages. These 2D class average images were carefully realigned to ensure that in all images the helix was parallel to the y axis, using SPIDER 50 , they were then projected to one dimension (1D) using Bsoft 51 . Radial density for each class average was plotted using Prism 8 (GraphPad), and fibril diameters were estimated as the distance between the minima on each side of the helix axis. Based on this 2D classification analysis, a dataset of fibril images that gave sharp class averages was selected. 3D classification was performed with k = 15, leading to the definition of nine classes of fibrils having diameters that reflected the full distribution of diameters identified by measurements of the radial density profiles. Inspection of the resulting reconstructions revealed that maps corresponding to the narrower class of fibril (Ø = 20-21.5 nm) could be described as left-handed 1-start helices with twist values of~-158.8°and axial rise values of 15-17 Å. The broader class of fibrils (Ø = 21.7-22.3 nm) were twofold symmetric about the helix axis and were processed as right-handed 2-start helices (twist =~40°, axial rise = 27-31 Å) with C2 symmetry imposed. 3D automated refinement led to the calculation of density maps for each class at resolutions ranging between 7.6 and 10.9 Å. As the reconstructions comprised an asymmetric unit that was a dimer, there was a twofold symmetry present that could not be accommodated within the helical reconstruction process. This was subsequently imposed on half-maps using UCSF Chimera 52 . Briefly, two copies of each half-map were opened, one was rotated 180°about the x axis (perpendicular to the helix axis), the fit in map function was then used to align the two maps and the density was averaged. Symmetrized half-maps were then imported into Relion for postprocessing analysis, resolution estimates ranged between 7.3 and 9.5 Å.
Reconstructed density maps were interpreted by docking of published coordinates for the FMDV 3D pol protein using UCSF Chimera 52 and visualised using UCSF ChimeraX 53 .
Replicon plasmids. The FMDV replicon plasmid, pRep-ptGFP, along with equivalent constructs containing the replication-defective GNN point mutation to the 3D pol polymerase, have been described previously 37 . In this replicon, the capsid protein-coding region has been replaced with a green fluorescent reporter protein.
Mutations were introduced into the pRep-ptGFP replicon 3D pol coding region by overlapping PCR site-directed mutagenesis. The sequences of primers are available on request.
FMDV replicon replication assays. To generate in vitro transcribed RNA for transfection, replicon plasmid DNA linearized with AscI (NEB, Ipswich, Massachusetts, USA) was used to setup T7 in vitro transcription reactions, as previously described 37 . T7 RNA transcripts were purified using a Zymogen RNA Clean and Concentrator-25 (Zymo Research, Irvine, California USA) according to manufacturer's instructions and the RNA quality was determined by MOPSformaldehyde agarose gel electrophoresis.
To assay replicon replication, BHK-21 cells seeded into 24-well tissue cultures plates were allowed to adhere for 16 h before transfection in duplicate with in vitro transcribed replicon transcripts using Lipofectin reagent as previously described 37 . Fluorescent reporter protein expression was monitored using an IncuCyte Zoom Dual Colour FLR (Essen BioSciences), within a 37°C humidified incubator, scanning hourly up to 24 h post-transfection. Images were captured and analysed using the associated software for fluorescent protein expression. Control transfections (untransfected and the 3D pol -GNN transfection to assess the level of input translation) were used to determine fluorescence thresholds and identify positive objects from background fluorescence. The number of positive cells per well was determined from the average of up to nine non-overlapping images per well.
Statistics and Reproducibility. The methods for statistical analysis and sizes of the samples (defined as n) are specified in both the results section and individual figure legends for all of the quantitative data. All values are presented as mean ± SEM with the indicated sample size where applicable. Statistical differences were determined using two-tailed unpaired t test and two-way analysis of variance (ANOVA) where appropriate. Statistics were performed using GraphPad Prism 8.
Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.


Does Protocatechuic Acid Affect the Activity of Commonly Used Antibiotics and Antifungals?

The aim of this study is to evaluate the efficiency of protocatechuic acid (PCA) in enhancing the commonly used drugs used to fight against nosocomial infection. These drugs are represented by routinely used antibiotics, synthetic chemotherapeutic agents with an antimicrobial spectrum, and antifungals. Three concentrations of PCA were added to 12 types of commercial disks used for antibiotic and antifungal susceptibility and tested against bacterial and yeast strains represented by Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. The results proved that PCA increased up to 50% of the antibacterial activity, especially that of levofloxacin against Staphylococcus aureus and Escherichia coli. These formulations will lead to new drug design ideas containing a smaller amount of antibiotics with the same effectiveness.

Introduction
Bacterial infections are a major challenge in modern medicine due to the well-known fact that many micro-organisms become resistant to antibiotics and antifungals over time [1], resulting in approximately 700,000 deaths each year [2]. As a result, drug resistance is becoming an Achilles heel in modern medicine [3].
However, despite reports of clinical failure of antibiotics and antifungals with in vitro resistance detection, they are still in use today [4]. One of the implications is that patients are at risk of developing multidrug resistance [5]. Even the broad-spectrum cephalosporins of the third generation, such as ceftazidime and cefotaxime, should be used sparingly due to their reported drug resistance [6]. Apart from that, in clinical practice, antibiotics of beta-lactam class, including amoxicillin, cephalosporins, and carbapenems, used to treat Gram-negative bacterial infections, are not recommended to be administered alone due to the difficulty of resistance mechanisms (a metallo-lactamase enzyme produced by Gram-negative bacteria can inhibit beta-lactam antibiotics) [7].
Recently, the group of Prof. Andersson identified resistance against nitrofurantoin [3]. Nitrofurantoin is a synthetic derivative of nitrofuran antibiotics that inhibits DNA, RNA, and cell wall protein synthesis and is used against urinary tract infections [8][9][10].
Due to the extensive use of trimethoprim antibiotics [11,12] in the treatment of urinary tract infections, antimicrobial resistance has increased, pushing researchers to pursue a deeper understanding of the mechanisms of resistance in order to improve the drug's future use [13]. A major source of morbidity in hospitals, nosocomial infections are caused by catheter-associated urinary infections, which are resistant to ampicillin (40-100%), cotrimoxazole (75-90%), tetracycline (66.7-86.6%), and ciprofloxacin (69%), as well as up to 71.4% resistant to norfloxacin [14]. Drug resistance has also emerged in the treatment of respiratory infections. In a recent study, the group of Sediana showed that in the treatment of respiratory infections with levofloxacin, 50% of 142 patients had high levels of resistance, 30.95% had intermediate levels of resistance, and 19.04% of patients were still sensitive to the antibiotic [15].
Besides antibiotics, antifungal drugs are also used to treat various mycoses. These can be polyene antifungal drugs (amphotericin B and nystatin), which bind to ergosterol, a component of the cell membrane of fungi, or azole antifungal drugs (ketoconazole, econazole, and fluconazole), which inhibit the conversion of lanosterol to ergosterol [16]. It is worth noting that amphotericin B, is the "gold-standard" antifungal for Candida infections and has been used in clinical practice for more than 40 years [17]. However, growing drug resistance and the negative side effects of antifungal medications limit their clinical uses [18][19][20]. Finally, expanding the usage of medicine requires its efficacy to be increased and its toxicity to be reduced.
In this regard, new approaches are needed to achieve effective therapy despite resistance, as well as to reduce the emergence of resistance, long-term persistence, or collateral sensitivity, which can increase the susceptibility of drugs by a factor of two to four [3].
To eliminate or reduce drug resistance, it is important to lower doses by using synergistic interactions with other compounds, such as natural antioxidants extracted from plants [21]. One such compound is protocatechuic acid (PCA), a phenolic compound found in medicinal plants, spices, and traditional Chinese herbal medicines exhibiting many important activities, such as antioxidant, antibacterial, and antiviral activities [22]. PCA is a widely distributed bioactive compound that it is found in many plants, such as Euterpe oleracea, Allium cepa, Daucus carota; mushrooms, such as Agaricus bisporus and Phellinus linteus; loquat fruit; honey; soybean; fruits of Ficus species; spices, such as Illicium verum, Melissa officinalis, Rosmarinus officinalis, and Cinnamomum aromaticum [23]; or medicinal plants, such as Ginkgo biloba, Cibotium barometz, and Stenoloma chusanum [22].
The PCA antioxidant action comes from the property of scavenging free radicals by donating hydrogen atoms or electrons [24], whereas the antibacterial activity is due to the action of decreasing lipid oxidation levels [25], and its antiviral property is due to the replication of the hepatitis B virus [26]. Our group also loaded unstable and insoluble PCA into magnetic cargo complexes under mild conditions using a simple and reproducible approach to increase its bioavailability and its continuous pharmaceutical effects, by releasing it for more than three days [27][28][29][30].
Within this context, the aim of this research is to test the synergistic effect of PCA and commonly used antibiotics/synthetic chemotherapeutic agents with antimicrobial spectra (such as amoxicillin/clavulanic acid, ceftazidime, gentamicin, nitrofurantoin, and cefotaxime) and antifungals (such as amphotericin B, nystatin, econazole, ketoconazole, cotrimoxazole, and fluconazole) against four different reference strains (Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27583, and Candida albicans ATCC10231) to fight nosocomial infections, as well as to aid in the development of drug switches to combat drug resistance.

Methods
The antimicrobial activity of the samples (commercial discs, PCA solutions, and their combinations) was determined by disk diffusion assay [31,32] against four different reference strains (Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27583, and Candida albicans ATCC10231) according to the recommendations of the disc manufacturer. All micro-organisms were stored at −80 • C in 20% glycerol. The bacterial strains were refreshed on Mueller-Hinton agar at 37 • C, and the fungal strain was refreshed on Sabouraud dextrose agar at 37 • C. Microbial suspensions were prepared with these cultures in sterile solution to obtain turbidity optically comparable to that of 0.5 McFarland standards. Volumes of 0.2 mL from each inoculum were spread onto Mueller-Hinton agar and Sabouraud dextrose agar. The sterilized paper discs were placed on the inoculated Petri plates. Aliquots of 15 µL of the tested PCA concentrations were added to the paper discs. To evaluate the antimicrobial properties, growth inhibition was measured under standard conditions after 24 h of incubation at 37 • C. All tests were carried out in triplicate to verify the results. After incubation, the diameters of inhibition zones were measured using Image J software [33]. The ratio of amplification of antibacterial/antifungal activity is defined as: % = (Izb − Iza)/Iha × 100%, where Iza and Izb are the average values (mm) of inhibition zone of the control drug and drug-PCA, respectively.

Statistical Analysis
Results of antibacterial activity are expressed as the mean ± standard deviation (SD) and were subjected to one-way analysis of variance (ANOVA), with p < 0.05 considered statistically significant, using XLSTAT Ecology software [34].

Results
In the present study, we examined the evolution of the inhibition of four reference strains (Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27583, and Candida albicans ATCC10231) when PCA formulations were administered with common antibiotics or other classes of antimicrobials ( Figure 1, Tables 1 and 2).

Discussion
Over the last decade, with respect to drug resistance, the research studies have considered the synergic effect produced by phytochemicals on antifungal medications [35]. One of these phytochemicals is PCA, found in medicinal plants, spices, and traditional Chinese herbal medicines in different concentrations. For example, in Ginkgo biloba, the content of free phenolic acids (protocatechuic and p-hydroxybenzoic acids) is 19.69 µg/g in fresh leaves and 345.32 µg/g in roots, where PCA is predominant [22]. Cibotium baromez, a traditional Chinese herbal medicine, presents a content of total phenolic acid of 3.73-6.16% in rhizome [36], whereas Hibiscus sabdariffa, with a well-known antiseptic effect, has a PCA content of 8.62% [37]. Allium cepa, which is recognized for its antifungal properties [22], presents a total flavonoids content (TFC) of 55.27 mg, with a total polyphenolic content (TPC) of 97.28 mg [38]. A product that is rich in PCA is represented by Acai palm (Eu-terpe oleracea), the oil of which has a 630 mg/kg PCA content [21]. PCA is also found in large quantities in various fruits and spices, such as: blackberries, 127 µmol/kg; mulberry, 119 µmol/kg; raspberry, 270 µmol/kg; strawberry, 112 µmol/kg; cranberry; gooseberry, 405 µmol/kg; dried dates, 320 µmol/kg; and star anise, 2.090 µmol/kg [39].
PCA is known in the literature to produce antibacterial effects due to its ability to inhibit bacterial growth. Inhibitory mechanisms of phenolic acids with respect to bacterial growth are nonspecific that can involve various modes of action, such as destabilization and permeabilization of the bacterial cytoplasmatic membrane; inhibition of virulence factors, such as enzymes and toxins, directly altering microbial metabolism; and inhibition of bacterial biofilm formation [40][41][42]. The main factor leading to these inhibitory actions is considered to be the hydroxyl groups of phenolic compounds, which can interact with the cell membrane of bacteria and damage the structures of the cell membrane [43]. Plant phenolic compounds have been considered powerful antibacterial agents, particularly for preventing the formation of biofilms by producing changes in the bacterial surface, reduction in exopolysaccharide production, and interference with cell communication [40,44,45]. Additionally, certain phenolic compounds function by a different mechanism than the known antibacterial agent. Different combinations may enhance or facilitate an antibiotic's engagement with its target inside the bacterial cell. Because lower dosages of both medicines can be utilized, the synergy can be used to broaden the antibacterial range, prevent the emergence of micro-organisms resistant to antibiotics, and minimize toxicity [45]. Furthermore, it has been reported that phenolic acids might damage the cytoplasmic membrane's structure, resulting in a loss of integrity and eventual cell death [46]. Lower antibiotic doses would be required because the compounds in the extract, at sub-inhibitory concentrations, would facilitate the antibiotic's entry into the cell cytoplasm, allowing fluoroquinolones, tetracycline, and chloramphenicol, which have their sites of action inside the bacterial cell, to enter more easily [47].
In this regard, several cases of phytochemicals improving the activity of the antibiotics have been presented in the literature. Jayaram et al. [48] studied the activity and interactions of antibiotics and phytochemicals against Pseudomonas aeruginosa in vitro. They tested seven different antibiotics (ciprofloxacin, tetracycline, ceftazidime, sulfamethoxazole, polymyxin B, trimethoprim, and piperacillin) in combination with six phytochemicals (protocatechuic acid, ellagic acid, gallic acid, berberine, rutin, and myricetin), and the results indicated that pairs of active principles, such as PCA-sulfamethoxazole, ellagic acid-sulfamethoxazole, and gallic acid-sulfamethoxazole have synergistic effects against Pseudomonas aeruginosa. In our study, the most promising result was achieved in the case of CTX and LEV in combination with PCA 1 , with their activities were increased by 13.59% and 15.25%, respectively, against the same bacterial strain.
Another study on antibiotic-phytochemical effects, was carried out by Kyaw, BM et al. [49], in which fusidic acid, rifampicin, cefotaxime, vancomycin, and ofloxacin were used in combination with six phytochemicals (tannic acid, quercetin, gallic acid, caffeic acid, eugenol, and menthone) against Staphylococcus aureus. The results indicated that tannic acid was synergistic with cefotaxime, rifampicin, fusidic acid, and minocycline, whereas quercetin was synergistic with fusidic acid, minocycline, and rifampicin. In this regard, our results show that the combinations of PCA 1 and LEV or GEN drugs exhibited a 55.98% and 20.55%, boost in activity against the same strains, respectively. This is a preliminary study demonstrating that simple combinations of well-studied substances, such as regularly used antibiotics and PCA, can be more effective than the pure antibiotic. We aim to use the promising drug-PCA combinations to generate nanoformulations consisting of magnetically driven therapeutic agents with the goal of lowering antibiotic dosages while retaining efficacy. These findings will have a positive impact on the development of novel pharmaceutical formulations as weapons in the fight against antibiotic resistance in bacteria.

Conclusions
This in vitro study proves that PCA addition can significantly improve the antibacterial activity of commonly used drugs, such as levofloxacin, nitrofurantoin, and cotrimoxazole, against both Gram-positive and Gram-negative bacterial strains by up to 50%. This is the first report on the possible beneficial effects of PCA in the administration of the drugs presented herein. Considering the results presented in this brief report, further studies are needed to understand the detailed aspects of the extent and the manner in which PCA and antibiotics/synthetic chemotherapeutic agents with a broad antimicrobial spectrum influence each other. However, these formulations will lead to new drug design ideas that contain a smaller amount of antibiotics with the same effectiveness.